Interpretive Summary: The Beltsville Sperm Sexing Technology is a well established procedure for pre-selecting the sex of offspring in most mammalian species. One of the characteristics of the procedure has been that sperm numbers sorted per hour was low, around 350,000 sperm. This manuscript illustrates the greater numbers of sperm that can be sorted per hour using the new high speed sorting technology that was recently made available. Upwards of 5 million sperm can be sorted per hour when using our new orienting nozzle adapted to high speed sorting. This is a 10 to 15 fold improvment in sexed sperm production per unit time. Sperm were sorted in this study to utilize with in vitro fertilization of pig eggs obtained from an abbatoir. The fertilized eggs were then developed to embryos and transferred to recipient females. Nine sows farrowed a total of 57 pigs. The three litters comprising the control group averaged 7.8 pigs per litter and were present in equal male and female numbers. Six litters were born from recipients that had received embryos produced from X bearing sperm. A total of 34 pigs were born, all were female except one (97%). Litter size was 5.8 for the sexed pigs. These data demonstrate the accuracy and feasibility of using sexed semen for the pig to produce litters predominantly of one sex or the other. Scientists will be implementing the high speed sorting systems so that more flexibility can be gained in using sexed semen in practical artificial insemination situations.

Technical Abstract:
The objectives of the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high speed sorting. In order to improve the IVF system with sorted sperm two fertilization media were tested (FERT- A vs. FERT-B). Fertilization with unsorted and sorted sperm resulted in higher cleavage rates when FERT-B was used as a fertilization medium (p<0.05). Additionally, cleavage rates were significantly higher (p<0.05) after fertilization with sorted sperm vs. unsorted semen, independent of FERT-A or FERT-B. Cytogenetic analysis of ova showed that more oocytes remained in Metaphase 2 when unsorted sperm were used for fertilization as compared to IVF with sorted semen. However, differences (p<.05) were found between IVF with FERT-A and unsorted sperm vs. FERT-A or FERT-B with sorted sperm. Polyspermic fertilization rates did not differ between groups. A total of fifty-seven pigs were born from 9 litters. Six litters from sexed sperm (X-sorted) produced 33 females and one male (97%). Three litters from control transfers produced 23 pigs, 12 of which were male (52%). The sex ratio of the offspring was predicted based on the reanalysis of the sorted sperm for DNA content. These studies demonstrate the effectiveness of the Beltsville Sperm Sexing Technology in combination with in vitro maturation, in vitro fertilization and embryo transfer to produce litters of pigs of predetermined sex.